Abstract Periodontal disease affects millions of individuals in the US alone and has been substantiated as a precursor to other debilitating systemic diseases, including cardiovascular disease, Alzheimer?s disease, rheumatoid arthritis, and adverse pregnancy outcomes. Our laboratories have shown that expression of certain microRNAs (miRNAs) are elevated in murine polymicrobial periodontitis. The current paradigm is that miRNAs are generally involved in fine-tuning gene expression. However, our in vitro studies have demonstrated that miR- 146a is a dominant miRNA that can be up-regulated 30 to 200+ fold during lipopolysaccharide stimulation and, more importantly, that this increase is sustained for days. We have demonstrated that miR-146a is a key regulator in endotoxin-induced tolerance and cross-tolerance using a monocyte/macrophage-based system. Similarly, we have demonstrated that miR-132 is a dominant miRNA in peptidoglycan-stimulated monocytes and can induce cross-tolerance. In the current proposal, these dominant miRNAs will be examined using in vitro and in vivo systems to critically determine their role in our established murine model of periodontitis with 4 major well-characterized periodontal pathogens (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum) and Streptococcus gordonii as early colonizer. The overall hypothesis is that these miRNAs are the dominant miRNAs regulating toll-like receptor (TLR)/IL-1-signaling pathways. Four Specific Aims are proposed. Specific Aim 1 will define the mechanistic role of these miRNAs, including mapping of target mRNAs, in primary human oral epithelial cells in reference to human monocytes. Specific Aim 2 will determine the expression kinetics of the dominant miRNA in mono- or time-sequential polymicrobial infection-induced periodontitis in mice and examine the relative effects of TLR2 and TLR4 using gene knockout mice. Specific Aim 3 will evaluate the relative contribution of these dominant miRNAs in this periodontitis model using specific miRNA knockout mice. Specific Aim 4 will investigate the association of these dominant miRNAs as biomarkers in gingival crevicular fluid and gingival tissues in chronic periodontitis and correlation with therapeutic outcomes. The long-term goal is to determine how extensively these dominant miRNAs can serve as functional biomarkers and they regulate innate immune response pathways contributing to periodontitis. In future studies, this mouse periodontitis model will become critical to help develop manipulation of these miRNA functions and/or the TLR pathway into novel therapeutics for periodontitis. Since innate immune response is known to play critical roles in many other diseases, our findings will likely be applicable to other chronic inflammatory and autoimmune diseases.